The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
A number of assays based on bioaffinity binding reactions or enzymatically catalyzed reactions have been developed to analyze biologically important compounds or their activity or their biological effect or its modulation from various biological samples (such as serum, blood, plasma, saliva, urine, faeces, seminal plasma, sweat, liquor, amniotic fluid, tissue homogenate, ascites, etc.), samples in environmental studies (waste water, soil samples), industrial processes (process solutions, products) and compound libraries (screening libraries which may comprise organic compounds, inorganic compounds, natural products, extracts of biological sources, biological proteins, peptides, or nucleotides, etc.). Some of these assays rely on specific bioaffinity recognition reactions, where generally natural biological binding components are used to form the specific binding assay (with biological binding components such as antibodies, natural hormone binding proteins, peptides, lectins, enzymes, receptors, single and double-stranded nucleic acids) or artificially produced binding compounds like aptamers, artificial nucleic acid mimics, genetically or chemically engineered antibodies (Lipovsek D, Pluckthun A. In-vitro protein evolution by ribosome display and mRNA display. J Immunol Methods. 2004; 290: 51-67; Pini A, Bracci L. Phage display of antibody fragments. Curr Protein Pept Sci. 2000; 1: 155-169; and Hoogenboom H R. Overview of antibody phage-display technology and its applications. Methods Mol Biol. 2002; 178: 1-37), moulded plastic imprint (molecular imprinting), other assays rely on activity or modulation of the activity of compounds present in sample or added into reaction (e.g. biologically active enzymes, chemical compounds with activity on biological molecules, enzyme substrates, enzyme activators, enzyme inhibitors, enzyme modulating compounds) and so on. Such assays generally rely on a label or a combination of multiple labels generating signals to quantitate the formed complexes after recognition and binding reaction. In heterogeneous assays a separation step (separations like precipitation and centrifugation, filtration, affinity collection to e.g. plastic surfaces such as coated assay tubes, slides or microparticles, solvent extraction, gel filtration, or other chromatographic systems, and so on) is generally required before e.g. the free or bound fraction of the label signal can be measured. In homogeneous assays the signal of the label or labels is modulated due to binding reaction or enzymatic activity or other measured effect and no separation step is needed before measurement of the label signal. Both in heterogeneous and homogeneous assays the measurement of the label signal from free or bound fraction of the label generally enables the calculation of the analyte or activity in the sample directly or indirectly, generally through use of a set of standards to which unknown samples are compared. Different binding assay methods have been reviewed recently in Principles and Practice of Immunoassay, 2nd ed., C. P. Price and D. J. Newman, eds., Palgrave Macmillan, Hampshire, UK, 2001; and The Immunoassay Handbook, 2nd ed. David Wild, ed., Nature Publishing Group, New York, N.Y., 2001.
Binders for Peptides and Polypeptides
Various types of proteins recognizing specific terminal peptide sequences can be produced artificially. Specific polyclonal and monoclonal antibodies (Köhler and Milstein, Nature (1975) 256: 495-497) against a wide range of chemical structures, including terminal sequences of peptides/polypeptides as well as specific peptide/polypeptide conformations, can be raised by immunizing mice or various other animals with suitable antigens. An example of a terminal sequence specific antibody is the monoclonal mouse antibody M1 raised against FLAG-Tag (DYKDDDDK) peptide epitope. In the presence of bivalent cation (preferably calcium), antibody M1 specifically binds to free N-terminus of the FLAG-peptide with high affinity, but has does not recognize the FLAG-peptide if there are one or more additional amino acids in the N-terminus of the peptide (Pricett et al., Bio Techniques (1989) 7: 580-589). The antibody MFS, in turn, is able to recognize a divalent cation-induced conformational change in myosin light chain 2 (Reinach and Fischman, J. Mol. Biol. (1985) 81:411-22).
Antibodies against terminal sequences or specific protein conformations can also be developed by selecting them from recombinant antibodies libraries (see e.g., Marks, et al., J. Mol. Biol. (1991) 222, 581-97., Knappik, et al., J. Mol. Biol. (2000) 296, 57-86; Söderlind, et al., Nat. Biotechnol. (2000) 18, 852-6) using, for example, phage display technique (Smith, Science 228 (2005), 1315-7). For example, recombinant antibody phage libraries were employed for development of the recombinant antibody Fab-fragment H2 which exclusively binds to active Ras protein, and not to inactive Ras (Horn et al., FEBS Lett. (1999) 463:115-20). Currently, several other protein frameworks (scaffolds) in addition to antibodies have been recruited for the development of recombinant protein binders against various targets. The binders based on scaffold proteins include, for example, anticalins (based on lipocalin structures; Skerra, Rev. Mol. Biotechnol, (2001) 74: 257-275), trinectins (derived from a fibronectin III domain; Xu et al., Chem. Biol. (2002) 9: 933-942), and affibody molecules (engineered from the Z domain of protein A; Nord et al., Nat. Biotechnol., (1997), 15: 772-777) as well as DARPins (designed on ankyrin repeat protein framework: Binz et al Nat. Biotechnol. (2004) 22:575-82). More examples of the scaffolds can be found, for example, in the review article by Binz and Pluckthun (Curr. Opin. Biotechnol. (2005) 16: 459-469). The scaffold protein based binder libraries can be subjected for selections and screening procedures in order to develop binders against terminal sequences or conformational epitopes in peptides and polypeptides. It is likely that in the future yet another protein folds will be recruited for development of binder molecules. It is also possible to generate completely new proteins folds with specific binding activities using, for example, by exon shuffling approach (Riechmann and Winter, Proc Natl Acad Sci USA. (2000) 97: 10068-73). Owing to the progress in the field of computer-assisted structural modelling of proteins, it has become possible to produce specific binder proteins against various targets by designing binding sites in silico into a suitable framework structure followed by introduction of the corresponding mutations in practice. (Looge et al., Nature (2003) 423:185-90). Various protein folds can act as a framework for in silico design approaches.
A large number of natural proteins interact with each other in a conformation specific manner. For example, spectrin EF-hands undergo a major conformational change upon calcium binding from a ‘closed’ to an ‘open’ state allowing protein-protein interaction (Trave et al., EMBO J. (1995) 14:4922-31). Again, several natural proteins specifically recognize terminal peptide sequences (either N- or C-terminal) in other proteins. These proteins or their domains responsible for the binding provide a potential source of binder molecules for recognition of specific conformation or terminal peptide segments. PDZ domain and PYX, for example, are specialized for recognition of C-terminal peptide sequences (Chung et al., (2002) Trends Cell Biol. 12:146-50). Another protein type binding to C-terminal sequences are known as 14-3-3 proteins, which can bind to specific phosphorylated C-terminal sequences (Coblitz, et al. FEBS Lett. (2006) 580:1531-5). N-terminal sequences in endogenous proteins are, in turn, specifically recognized by certain N-end rule pathway related proteins as a part of a process targeting proteins for degradation. In prokaryotes, CIpS protein binds to the degradation signal that comprises N-terminal destabilizing residues (Erbse et al., Nature (2006) 439:753-6), while, in eukaryotes, the N-terminal degradation signal of the N-end rule is recognized by N-recognin proteins (Varhaysky, Proc. Natl. Acad. Sci. (1996) 93, 12142-12149). If necessary, various mutagenesis methods can be employed for modulation of the binding properties (such as the specificity and affinity) of terminal peptide recognizing natural proteins (see, for example, Skelton et al., J Biol. Chem. (2003) 278:7645-54).
Aptamers are nucleic acids based binder molecules, which primarily consist of natural nucleic acids (DNA and RNA) and can also contain artificially introduced chemical moieties such as non-natural nucleotides. Specific aptamers against various target molecules can be systematically produced by using, for example, a technique called systematic evolution of ligands by exponential enrichment (SELEX; Tuerk and Gold, Science (1990) 249: 505-510), where nucleic acid molecules are, first, selected for binding to target and then amplified by using an enzymatic reaction. Until today, specific aptamers have been developed for a wide array of target molecules ranging from small molecular compounds to large macromolecules such as proteins (Proske, et al., Appl Microbiol Biotechnol. (2005) 69:367-74), and it should be possible to produce aptamers that specifically bind either to N- or C-terminal peptide sequences or to specific peptide/polypeptide conformations. For example, aptamers that bind selectively to PrPC form of the prion protein and not PrPSc form have been developed (Takemura et al., Exp Biol Med (Maywood) (2006). 231:204-14).
Binders for Carbohydrates
Specific antibodies against various carbohydrate structures can be produced by immunization or by selecting/screening binders from recombinant antibody libraries (see, for example, Sakai et al., Biochemistry (2007) 46:253-62). Various scaffold proteins based libraries as well as aptamer libraries (Masud at al, Bioorg Med. Chem. (2004) 12:1111-20) can also be employed for the development of carbohydrate binders. In addition to the artificially developed binders, there are a number a natural proteins that have capability to recognize and bind specific carbohydrate structures. For example, lectins form a diverse group of proteins that have in common their ability to specifically recognize certain carbohydrates (Loris., Biochim Biophys Acta. (2002) 1572:198-208). Some lectins show discrimination between different terminal structures of carbohydrates (see, for example, Hirabayashi et al., Biochim Biophys Acta. (2002) 1572:232-54). The binding properties of the natural carbohydrate binder proteins can be modulated by means of protein engineering (Yabe et al., J Biochem (Tokyo) (2007) Epub ahead of print).
Binders for Nucleic Acids
The capability of a nucleic acid segment to specifically hybridize to another nucleic acid segment with complementary sequence is widely employed for detection of nucleic acid sequences of interest. The single stranded nucleic acid probes used for detection of a target nucleic acid sequence can be based either on natural nucleic acids, DNA or RNA, and their chemically modified derivatives as well as on artificial nucleic acid analogues such as peptide nucleic acids (PNA), locked nucleic acids (LNA) or morpholinos (Karkare and Bhatnagar, Appl Microbiol Biotechnol. (2006) 71:575-86). DNA has also property to form triple helix structures: A third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix, and this property can also be utilized for specific recognition of DNA sequences (see, for example, Ji et al., Genomics (1996) 31:185-92). Antibodies (Di Pietro et al., Biochemistry (2003) 42:6218-27) and apparently various scaffold protein as well as aptamers can also be utilized for specific recognition of DNA segments.
Fluorescence Resonance Energy Transfer
Fluorescence resonance energy transfer (FRET) (Förster, T. Intermolecular energy migration and fluorescence. Ann. Physik 1948; 2, 55-75.) (or Förster resonance energy transfer) describes an energy transfer mechanism between two fluorescent molecules or between a fluorescent and a non-luminescent molecule. A fluorescent donor is excited at its specific fluorescence excitation wavelength. By a long-range dipole-dipole coupling mechanism, this excited state is then nonradiatively transferred to a second molecule, the acceptor, which is luminescent and can emit at its specific emission wavelength, or the quencher, which is non-luminescent or luminescent. The donor returns to the electronic ground state. The mechanism is widely employed in biomedical research (reviewed by Selvin P R The renaissance of fluorescence resonance energy transfer. Nat Struct Biol 2000; 7: 730-734; and Lakowicz, J. Principles of fluorescence spectroscopy, 2nd edition. Plenum Press, New York, 1999).
The FRET efficiency is determined by the distance between the donor and the acceptor, the spectral overlap of the donor emission spectrum and the acceptor absorption spectrum, and the relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment. The FRET efficiency E depends on the donor-to-acceptor distance r with an inverse 6th order law defined byE=1/(1+(r/R0)6)with R0 being the Förster distance of this pair of donor and acceptor at which the energy transfer efficiency is 50%. The Förster distance depends on the overlap integral of the donor emission spectrum with the acceptor absorption spectrum and their mutual molecular orientation.Self-Quenched Fluorescent Oligomers and Oligomeric Substrates
Bio-oligomer derivatives, for example oligopeptide, oligonucleotide and oligosaccharide derivatives, containing both a fluorescent moiety and a quencher moiety covalently attached typically to different ends of the same oligomer molecule, are employed to measure hydrolysation or cleavage of the oligomer upon for example enzymatic or chemical activity. The hydrolysis and cleavage, resulting in increase in the distance between a fluorescent moiety and a quencher moiety, are accompanied by an increase in the fluorescence due to disruption of the intramolecular quenching of the fluorescent moiety. The spectral properties of the moieties do not necessarily need to be consistent with an energy transfer mechanism according to Förster requiring spectral overlapping between emission spectra of the fluorescent moiety (donor) and excitation spectra of the quencher moiety.
Self-quenched oligopeptide substrates, also called fluorogenic substrates, and their applications have been described e.g. by Lottenberg R, Christensen U, Jackson C M, Coleman P L Assay of coagulation proteases using peptide chromogenic and fluorogenic substrates. Methods Enzymol. 1981; 80: 341-61; and by Lew R A, Tochon-Danguy N, Hamilton C A, Stewart K M, Aguilar M I, Smith A L Quenched fluorescent substrate-based peptidase assays. Methods Mol Biol. 2005; 298: 143-150. The use of specific quenched fluorescent oligopeptide substrates provides a rapid and sensitive method to measure peptidase activity, and is readily adaptable to high-throughput screening of potential peptidase inhibitors. A high throughput assay based on a peptide labelled with both a fluorescent europium chelate and a quencher has been described by Karvinen J, Hurskainen P, Gopalakrishnan S, Burns D, Warrior U, Hemmila I. Homogeneous time-resolved fluorescence quenching assay (LANCE) for caspase-3. J Biomol Screen. 2002; 7: 223-231. The principle of a peptidase assay based on quenched fluorescent substrate is illustrated in FIG. 1. In an intact fluorescent substrate the fluorescent label is quenched by the quencher, but when a peptidase cleaves the substrate the distance between the fluorescent label and the quencher increases recovering the fluorescence of the fluorescent compound. The measured signal is increased upon cleavage of the substrate.
Cleavage of the peptide by caspase-3 separates the quencher from the chelate and thus recovers fluorescence of europium chelate. A similar assay is possible by using a long-lifetime fluorescent metal-porphyrin label (O'Riordan T C, Hynes J, Yashunski D, Ponomarev G V, Papkovsky D B. Homogeneous assays for cellular proteases employing the platinum(II)-coproporphyrin label and time-resolved phosphorescence. Anal Biochem 2005; 342: 111-119). Phosphorescent platinum(II) coproporphyrin label was evaluated for the detection of cellular proteases by time-resolved fluorescence in homogeneous format. An octameric peptide containing the recognition motif for the caspase-3 enzyme was dual labelled with a new maleimide derivative of phosphorescent platinum(II) coproporphyrin label and with the non-luminescent quencher dabcyl. Donor-acceptor energy transfer and fluorescence quenching based assays have been described also for other enzymes: a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades (Karvinen J, Laitala V, Makinen M L, Mulari O, Tamminen J, Hermonen J, Hurskainen P, Hemmila I. Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase. Anal Chem 2004; 76: 1429-1436).
A cleavage assay can also be constructed using e.g. a terbium-chelate donor labelled streptavidin and using a biotinylated peptide substrate containing dabcyl as non-luminescent quencher or fluorescein as a luminescent acceptor at the other end of the peptide sequence. A similar cleavage assay using europium-chelate and donor labelled biotinylated peptide and streptavidin conjugate of XL665 luminescent acceptor is described in Kennedy M E, Wang W, Song L, Lee J, Zhang L, Wong G, Wang L, Parker E. Measuring human beta-secretase (BACE1) activity using homogeneous time-resolved fluorescence. Anal Biochem. 2003; 319: 49-55.
The principle of an assay with non-luminescent quencher is illustrated in FIG. 2, where the intact peptide contains both biotin and quencher moieties and is capable to bind to a fluorescent conjugate of streptavidin and quenches the fluorescence of the fluorescent label. When the peptide is cleaved the biotin and quencher moieties are separated and the quencher label is unable to bind to streptavidin and the fluorescence of the fluorescent label is not affected. Thus, the measured signal is increased upon cleavage of the substrate, because the cleavage prevents the quenching of the fluorescent label. The concentration of the fluorescent conjugate of streptavidin must be carefully adjusted because an excess of it results in a significant increase in the background signal.
FIG. 3 illustrates an assay based on a luminescent acceptor, where the substrate contains both biotin and acceptor moieties and is capable to bind to a donor conjugate of streptavidin. The sensitized acceptor emission is dependent on the proximity of donor and acceptor and only the acceptor present in an intact substrate is able to bind to streptavidin. Upon cleavage of the substrate the measured signal is decreased. The donor conjugate of streptavidin can be used in excess because signal without significant increase in the background signal. This method is used by Invitrogen (Carlsbad, Calif.; www.invitrogen.com) in their Lanthascreen concept based on terbium-chelate labelled streptavidin and biotinylated substrate labelled with fluorescein (http://www.invitrogen.com/-downloads/F-13279_LanthaScreen_Poster.pdf). The time-resolved FRET value is determined as a ratio of the FRET-specific signal measured with a 520 nm filter to that of the signal measured with a 495 nm filter, which is specific to terbium-chelate.
Fluorescence quenching assay based on an electrochemiluminescent label and luminescence quenching based on energy transfer is described in Spehar A M, Koster S, Kulmala S, Verpoorte E, de Rooij N, Koudelka-Hep M. The quenching of electrochemiluminescence upon oligonucleotide hybridization. Luminescence 2004; 19: 287-95. Interaction between electrochemically excited Ru(bpy)32+ and Cy5 in a hybridization assay on a chip was studied. The 3′ end of an oligonucleotide was labelled with Ru(bpy)32+ and the 5′ end of a complementary strand with Cy5. Upon the hybridization, the electrochemiluminescence (ECL) of Ru(bpy)32+ was efficiently quenched by Cy5 with a sensitivity down to 30 nmol/l of the Cy5-labelled complementary strand. The quenching efficiency is calculated to be 78%.
Quantitative 5′-nuclease based polymerase chain reaction assay (TagMan; Applied Biosystems, Foster City, Calif.) is a nucleic acid sequence detection method wherein a single-stranded self-quenching oligonucleotide probe, containing both a fluorescent moiety and a quencher moiety, is cleaved by the nuclease action of nucleic acid polymerase upon hybridisation during nucleic acid amplification (Lie Y S, Petropoulos C J. Advances in quantitative PCR technology: 5′ nuclease assays. Curr Opin Biotechnol. 1998; 9: 43-48; and Orlando C, Pinzani P, Pazzagli M. Developments in quantitative PCR. Clin Chem Lab Med. 1998; 36: 255-269).
Molecular beacons are single-stranded oligonucleotide hybridization probes that form a stem-and-loop structure (Tan W, Wang K, Drake T J. Molecular beacons. Curr Opin Chem. Biol. 2004; 8: 547-553; and Tan W, Fang X, Li J, Liu X. Molecular beacons: a novel DNA probe for nucleic acid and protein studies. Chemistry 2000; 6: 1107-1111). The loop contains a nucleic acid probe sequence that is complementary to a target sequence, and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence. A fluorescent moiety is covalently linked to the end of one arm and a quencher is covalently linked to the end of the other arm. Due to the proximity of a fluorescent moiety and a quencher moiety molecular beacons do not fluoresce when they are free in solution. However, when they hybridize to a complementary nucleic acid strand containing a target sequence they undergo a conformational change increasing the distance between fluorescent moiety and the quencher moiety that enables the probe to fluoresce. In the absence of a complementary target sequence, the beacon probe remains closed and there is no fluorescence due to intramolecular quenching.
Selective cleavage of internucleotide bonds of self-quenched single-stranded oligonucleotide probes, which contain one or more ribonucleotides, by RNase H upon double-stranded helix formation subsequent to hybridisation to target is another method of target sequence detection (Rizzo J, Gifford L K, Zhang X, Gewirtz A M, Lu P. Chimeric RNA-DNA molecular beacon assay for ribonuclease H activity. Mol Cell Probes 2002; 16: 277-283). Yet another method is to use a self-quenched single-stranded oligonucleotide cycling probe, which is cleaved by a double-stranded selective exonuclease upon hybridisation to target sequence. Examples of nuclease-based assays are found in e.g. Till B J, Burtner C, Comai L, Henikoff S, Nucleic Acids Res. 2004; 32: 2632-2641.
Self-quenched fluorescent probes are also used to monitor nucleic acid amplification process in a thermal cycler; for example in quantitative polymerase chain reaction the amount of fluorescence at any given cycle, or following cycling, depends on the amount of specific product. The self-quenched single-stranded fluorescent probes, for example molecular beacons or Taqman probes, bind to the amplified target following each cycle of amplification and the resulting signal upon hybridisation, and in case of Taqman probes upon cleavage, is proportional to the amount of the amplified oligonucleotide sequence. Fluorescence is measured during each annealing step when the molecular beacon is bound to its complementary target or after elongation step when the Taqman probe is cleaved. The information is then used during quantitative PCR or RT-PCR (reverse transcriptase PCR) experiments to quantify initial copy number of amplified target nucleic acid sequence. For endpoint analysis, PCR or RT-PCR reactions containing molecular beacons can be run on any 96-well thermal cycler and then read in a fluorescence reader.
Fluorescent oligosaccharide substrates and their use in fluorescence quenching assay has been described in Cottaz S, Brasme B and Driguez H, A fluorescence-quenched chitopentaose for the study of endo-chitinases and chitobiosidases. Eur. J. Biochem. 2000; 267: 5593-5600.
Non-fluorescent acceptor labels and their use in fluorescence quenching assays with short-lifetime fluorescent dyes have been described e.g. in U.S. Pat. No. 6,828,116.
Ribonuclease detection using dual-labelled quenched fluorescent oligonucleotide containing both short-lifetime fluorescent dye and non-luminescent acceptor has been described in US 2004/0137479.
Fluorescent quenching assay for protein kinase based on fluorescent labelled substrate and phosphate specific binder labelled with non-luminescent acceptor is described in US 2004/024946.
Fluorescence quenching assays based on both fluorescent streptavidin-coated microspheres and conjugates of small-molecule fluorescent dyes in combination with both non-luminescent acceptor dye and quencher polymer have been described in US 2003/0054413.
Fluorescence quenching assay based fluorescent streptavidin-coated microsphere and biotinylated non-luminescent acceptor labelled protease substrate for measurement of protease activity has been described in US 2005/0014160.
Protease activity assay based on dual-labelled fluorescent protein substrate containing binding moiety for purification and separation is described in US 2005/0214890.
In all of the aforementioned examples a fluorescent moiety or a fluorescent compound (donor) is used in combination with either non-luminescent compound (quencher) or luminescent compound (acceptor), respectively, and the donor compound is capable of transferring energy either to a quencher or to an acceptor, respectively, said energy transfer being dependent on the distance between the donor and quencher or acceptor.
In all cases the donor is excited directly by light or electrochemically and, in case of a non-luminescent acceptor, it's the donor's own light emission (fluorescence) is measured or in case of luminescent acceptor, the sensitized emission of an acceptor (originating from energy transfer) is measured.
Advantages of fluorescence quenching based assays and use of quenched fluorogenic substrates to improve the fluorescence-based enzyme assays have been described e.g. by Johansson, M K, Choosing reporter-quencher pairs for efficient quenching through formation of intramolecular dimers. Methods Mol. Biol. 2006; 335: 17-29; and Yang Y, Babiak P and Reymond J L, Low background FRET-substrates for lipases and esterases suitable for high-throughput screening under basic (pH 11) conditions. Org Biomol Chem. 2006; 4: 1746-54.
Homogeneous Bioassay Technologies
Homogeneous assay methods (Ullman E F, J Chem Ed 1999; 76: 781-788; Ullman, E F, J Clin Ligand Assay 1999; 22: 221-227) based on photoluminescence have received much attention, since several types of physical and chemical interactions can be employed to modulate the emission of photoluminescent labels due to formation of specific immunological complexes. The commonly employed methods are based on polarization of the emitted light or nonradiative energy-transfer between two photoluminescent compounds or between a photoluminescent and a non-luminescent compound (Hemmilä I, Clin Chem 1985; 31: 359-370). Fluorescence properties of two fluorescent compounds were employed in a homogeneous immunoassay in late 1970' s when Ullman et al. demonstrated, that fluorescence energy transfer between a fluorescein donor and tetramethylrhodamine acceptor pair could be employed to construct both competitive and non-competitive immunoassays (Ullman E F et al. J Biol Chem 1976; 251: 4172-4178; Ullman E F & Khanna P L, Methods Enzymol 1981; 74: 28-60). The energy transfer was measured from decrease in the fluorescence of the donor, which limited further improvements in sensitivity. Increase in the fluorescence of the acceptor was not practicable, since only a little increase in a sensitized acceptor emission could be observed over autofluorescence, light scattering or absorbance of biological sample matrices and the direct emission of the donor at acceptor-specific wavelength.
Many compounds and proteins present in biological fluids or serum are intrinsically fluorescent, and the use of conventional fluorophores leads to serious limitations of sensitivity (Wu P and Brand L, Anal Biochem 1994; 218:1-13). Another major problem with homogeneous fluorescence techniques is the inner filter effect and the variability of the optical properties of a sample. Sample dilution has been used to correct this drawback, but always at the expense of analytical sensitivity. Feasibility of fluorescence energy transfer in immunoassays was significantly improved when fluorescent lanthanide cryptates and chelates with long-lifetime emission and large Stokes' shift were employed as donors in the 1990' s (Mathis G, Clin Chem 1993; 39: 1953-1959; Selvin P R et al., Proc Natl Acad Sci USA 1994; 91: 10024-10028; Stenroos K et al., Cytokine 1998; 10:495-499; WO 98/15830; U.S. Pat. No. 5,998,146; WO 87/07955). Feasibility of the label technology in dissociation reactions, e.g. cleavage assays has also been described (Karvinen J et al., J Biomol Screen 2002; 7: 223-231).
Time-resolved fluorescence detection of sensitized emission allowed elimination of autofluorescence (Soini E and Kojola H Time-resolved fluorometer for lanthanide chelates—a new generation of nonisotopic immunoassays. Clin Chem 1983; 29: 65-68). Dual signal ratio measurement (U.S. Pat. No. 5,527,684; Mathis, G, Clin Chem 1993; 39: 1953-1959) corrected the variability of optical properties of the sample in homogeneous assay. Fluorescence of the compounds and proteins present in biological fluids has a short lifetime and the use of long-lifetime labels combined with time-resolved detection of the sensitized (prolonged lifetime) acceptor emission allowed minimization of the assay background and improved signal to background ratio. The variability of absorption of excitation light at 337 nm was corrected by measuring the emission of the donor at 620 nm and using the ratio of the energy transfer signal at 665 nm and the emission at 620 nm to generate a quantity that is independent of the optical properties of the serum sample. Homogeneous time-resolved FRET based bioaffinity assays using long-lifetime fluorescent nanoparticles have been described in WO 02/044725 and by Kokko L, Sandberg K, Lövgren T and Soukka T, Europium(III) chelate-dyed nanoparticles as donors in a homogeneous proximity-based immunoassay for estradiol Anal Chim Acta 2004; 503: 155-162. In the latter publication it is described that multiple lanthanide chelates inside a single particulate can participate simultaneously in energy transfer to a single acceptor. However, still only a small part of the lanthanide chelates inside the entire particulate can participate in an energy transfer to a single acceptor and thus the entire fluorescence of a particulate label cannot be quenched by a single acceptor moiety. The same problem is also encountered when lanthanide chelates are incorporated in a solid phase.
Separation-free assay technologies based on confocal detection of photoluminescent labels bound on particulate carriers have been introduced as an alternative to real homogeneous assays (Saunders G C et al., Clin Chem 1985; 31:2020-2023; Frengen J et al., Clin Chem 1993; 39:2174-2181; Fulton R J et al., Clin Chem 1997; 43:1749-1756). In recent years, the technology has been developed, and some novel carrier-based immunoassays can be considered as homogeneous assays, since they are practically similar to perform (Hänninen P et al., Nat Biotechnol 2000; 18:548; U.S. Pat. No. 5,891,738; Schaertl S et al., J Biomol Screen 2000; 5:227-238), although the actual signal of the label is not modulated, but the unbound labelled component is spatially excluded from measurement. These assays are otherwise comparable to homogeneous assays, but measurement is relatively slow, since carrier particles have to be either actively scanned or passively diffuse to a focal point, and a signal associated to several carrier particles is required for reliable measurement (Waris M E et al., Anal Biochem 2002; 309: 67-74). To avoid sterical hindrance in binding at least one of the labels, preferably both labels of a label-pair should be of small molecular size.
In most of the conventional homogeneous fluorescence assay technologies, the performance has still severe limitations: the sensitivity is limited by interferences from matrix components and optical properties of matrices, e.g. urine, saliva, serum, plasma or whole blood, to fluorescence yield and level of background, and by the attainable degree of fluorescence modulation, e.g. quenching, enhancement, energy transfer or polarization (Hemmilä I, Clin Chem 1985; 31: 359-370). In practice, only wavelengths in the range 600 to 1100 nm, or more preferably in the near infrared, in a wavelength range 650 to 950 nm, are practicable when a whole blood sample is employed (Chance B, Photon Migration in Tissues, pp. 206; Kluwer Academic/Plenum Publishers, 1990, New York).
Homogeneous luminescence-based whole-blood assay based on FRET and up-conversion photoluminescence is described in WO 2004/086049. Upconverting phosphors and their application as donors in FRET-based assays is described in WO 98/43072. Both the excitation and the measurement of sensitized acceptor emission have to be performed at far-red and infrared wavelengths where the sample is transparent, in this case at wavelengths 900-1000 nm and approximately 580-640 or 690-750 nm. Upconversion-FRET based assay performed in whole blood has been described by Kuningas K, Pakkila H, Ukonaho T, Rantanen T, Lovgren T and Soukka T., Upconversion fluorescence enables homogeneous immunoassay in whole blood. Clin Chem. 2007; 53:145-146.
U.S. Pat. No. 6,037,130 describes wavelength shifting probes comprising two conventional fluorescent molecules, which are both covalently attached to the probe, and a quencher, wherein energy-transfer excited emission of the second fluorescent molecule upon excitation of the first fluorescent molecule is measured.
US 2005/0170442 describes the use of a cleavable acceptor and biotin labelled peptide, a cleavage reaction inhibited by phosphorylation of a peptide, and an assay method based on energy transfer from streptavidin labelled with donor to the acceptor in presence of phosphorylation.
FRET-based hybridization assays utilizing multiple donors and donor-to-donor energy transfer are described in EP1067134; both donor and acceptor labelled oligonucleotides hybridize to third complementary target oligonucleotide enabling energy transfer from donor to acceptor. In the reaction mixture, a fourth quencher labelled oligonucleotide, complementary to acceptor labelled oligonucleotide, can be present.
Dual-FRET-probe based assay concept is described in WO 03/000933; in this application two molecular beacon-type quenched probes (one containing donor and quencher, the other acceptor and quencher) are hybridized next to each other to third complementary target oligonucleotide and energy transfer between donor and acceptor is enabled.
Hairpin-forming oligonucleotide probe or primer can be labelled with donor and acceptor (energy transfer pair) as well as quencher as described in WO 00/06778, U.S. Pat. No. 6,768,000 and also in US 2007/0077588. In the presence of a second complementary target oligonucleotide the distance between acceptor and quencher is increased, which enables energy-transfer excited acceptor emission.
Formation of a FRET-pair through ligation of two oligonucleotide probes, one labelled with donor and the other with acceptor, is described by Abe H and Kool ET (2006), Flow cytometric detection of specific RNAs in native human cells with quenched autoligating FRET probes, Proc Natl Acad Sci USA, 103: 263-8.